Download Competence

微生物遺傳與生物技術
(Microbial Genetics and
Biotechnology)
金門大學
食品科學系
何國傑 教授
Transformation
DNA exchange among bacteria
• DNA can be exchanged among bacteria in three
ways:
1. Conjugation – a plasmid or other selftransmissible DNA element transfers it self
and sometimes other DNA into other bacterial
cell.
2. Transduction – a phage carries DNA from
one bacterium to another.
3. Transformation – cells take up free DNA
directly from their envirnment.
Naturally transformable bacterium
• Most types of cells cannot take up DNA efficiently unless
they have been exposed to special chemical or
electrical treatments to make them more permeable.
1. Naturally transformable bacterium (or naturally
competent bacterium) – They can take up DNA from
the environment without requiring special treatment.
2. About 40 species have been found to be naturally
competent or transformable.
3. Bacillus subtilis, Streptococcus pneumoniae,
Haemophilus influenzae, Neisseria gonorrhoeae,
Helicobacter pylori, Acinetobacter baylyi,
and some species of marine cyanobacteria.
Artificially induced competence
•
Bacteria can be sometimes be made competent by
certain chemical treatments or DNA can be forced into
bacteria by a strong electric field in a process called
electroporation.
1. Treatment with calcium ions.
(1) Chemically induced transformation is usually
inefficient, and only a small percentage of the
cells are ever trnasformed.
(2) Accordingly, the cells must be plated under
conditions selective for the transformed cells.
(3) Therefore, the DNA used for the transformation
should contain a selectable gene such as one
encoding resistance to an antibiotic.
Artificially induced competence
2. Electroporation
(1) The bacteria are mixed with DNA and briefly
exposed to a strong electric field.
(2) The bacteria must first be washed extensively in
buffer with very low ionic strength such as distilled
water. The buffer usually also contains a nonionic
solute such as glycerol to prevent osmotic shock.
(3) The brief electric field across the cellular
membranes might create artificial pore of H2O lined
by phospholipid head groups. DNA can pass
through these temporary hydrophilic pores.
(4) Electroporation requires specialized equipment.
Discovery of transformation
1. In 1928, Fred Griffith found that one form of the
pathogenic pneumococci (now called Streptococcus
pneumoniae) could be mysteriously “transformed” into
another form.
Discovery of transformation
2. Griffith made a conclusion that the dead pathogenic
bacteria gave off a “transforming principle” that changed
the live nonpathogenic rough-colony-forming bacteria
into the pathogenic smooth-colony form.
3. Later, other researchers did an experiment in which they
trnasformed rough-colony-forming bacteria into the
pathogenic smooth-colony form by mixing the rough
forms with extracts of the smooth forms in a test tube.
4. About 16 later after Griffith did his experiment with mice,
Oswald Avery and his collaborators purified the
“transforming principle” from extracts of smooth-colony
formers and showed that it is DNA. Avery and his
colleagues were first to demonstrate that DNA, and not
protein or other factors in the cell, is the hereditary
material.
Competence
• The ability of some bacteria to take up naked
DNA from their environment.
• It is genetically programmed. Generally, more
than a dozen genes are involved, encoding both
regulatory and structural components.
• The general steps that occur in natural
transformation differ somewhat in Gram-negative
and positive bacteria.
• The followings are two examples for G – and
G + , respectively.
Competence
• The steps for DNA uptake
1. Binding of double-stranded DNA to the outer cell
surface of bacterium.
2. Movement of DNA across the cell wall and outer
membrane (no outer membrane in G + bacterium).
3. Degradation of one of the DNA strands.
4. Translocation of the remaining single strand of DNA
into cytoplasm of the cell across inner membrane.
5. Once in the cell, the single-stranded transforming
DNA might synthesize the complementary strand and
reestablish itself as a plasmid, stably integrate into
the chromosome, or degraded.
Competence
•
While the DNA uptake system of G + and G – bacteria have
features in common, they do seem to differ in certain important
respects.
1. There are many proteins involved in transformation in bacteria.
2. They are discovered on the basis of isolation of mutants that
are completely lacking in the ability to take up DNA.
3. The genes affected in the mutants were named com (for
competence defective).
(1) The com genes are organized into several operons.
(2) The products of these, including the comA and comK
operons, are involved in regulation of competence.
(3) Others, including the products of genes in the comE, comF,
and comG operons, become part of the competence
machinery in the membrane that takes DNA up into the
bacteria.
(4) The genes in these operons are given two letters, the first for
the operon and the second for the position of gene in the
operon, ex., comFA is the first gene of the comF operon.
Steps in natural transformation
• ComEA encoded by the first gene of the comE operon,
binds directly extracellular double-stranded DNA.
• The comF genes encode proteins that translocate the
DNA into the cell.
ComFA is an ATPase that may provide the energy for
translocation of DNA through the membrane (not shown).
• ComEA, ComEC, and ComFA form a sort of ATPDNA into the cell.
• The genes in the comG operon encode proteins that
might form a “pseudopilus” which helps move DNA
through the ComEC channel.
They might bind to extracellular DNA, perhaps acting
through the ComEA DNA-binding protein, and then retract,
drawing the DNA into the cell.
Steps in natural transformation
• The comE, comF and comG operons are all under the
transcrptional control of ComK, a transcriptio factor that
is itself regulated by ComA.
• Some of genes involved in the transformation process
are not designated as com, because such genes were
first discovered on the basis of their involvement in other
processes.
1. The nucA gene product makes double-strand breaks
in extracellular DNA. The free DNA ends become the
substrates for the competence proteins.
2. Other examples are single-stranded-DNA binding
protein (SSB), and RecA functions in the
recombination of transforming DNA with chromosome
DNA.
Steps in natural transformation
• The lengths of single-stranded DNA incorporated into the
recipient chromosome are about 8.5 to 12 kbbase on
cotransformation of genetic markers, and the
incorporation takes only few minutes to be completed.
• The proteins in shaded boxes are analogous in G + and
G – bacteria. ComEC of B. subtilis is an ortholog of
ComA protein of Neisseria.
• The DNA is shown running through the cell wall alonside
the pseudopilus (ComG in B. subtilis; PilE in G –
systems that are related to type II protein secretion
systems.
Natural transformation of Gram-positive
bacteria
• ComEA
binds
directly
extracellular
doublestranded
DNA.
• The comF genes encode proteins that translocate the DNA into
the cell.
• ComEA, ComEC, and ComFA form a sort of ATP-binding
cassette (ABC) transporter.
• The genes in the comG operon encode proteins that might form
a “pseudopilus” which helps move DNA through the ComEC
channel, and the ComECs retract,drawing the DNA into the cell.
Natural transformation of Gramnegative bacteria
4. PilQ
(secretion
proteins): 12 ~
14 copies
making the
pore through
the outer
membrane.
1. ComA protein of Neisseria is an ortholog of ComEC of B. subtilis.
2. The DNA is shown running through the cell wall alonside
the pseudopilus (ComG in B. subtilis; PilE in G – systems).
3. In most G – bacteria specific sequences are required for the
binding of DNA, so that these species usually take up DNA only
of the same species.
Natural transformation of Gramnegative bacteria
1. The competence systems of most G – bacteria are
very similar to type II secretion systems that
assemble type IV pili on the cell surface.
2. Type IV pili are long, thin hairlike appendages that
stick out from the cell and are used to attach cells to
solid surface.
3. While competence requires the protein that makes
up most of the pilus, i.e., the major pilin protein
(called PilE in Neisseria), some other minor pilin
proteins are required for competence but not for
pilus formation.
Natural transformation of Gramnegative bacteria
4. The competence of Helicobacter pylori, an opportunistic
pathogen involved in ulcer, bases on the type IV secretionconjugation systems.
(1) The proteins of type IV secretion-conjugation system is
similar to Vir conjugation proteins in Agrobacterium
tumefaciens.
(2) Type IV secretion-conjugation system can function as two
way DNA transfer systems, capable of moving DNA both
into and out of the cells.
(3) However, H. pylori has a bona fide type IV secretion system
that secrets proteins directly into eukaryotic cells. These
two systems are related, but they function independently of
each other and have no proteins in common.
▇ When the secretion systems, transformation systems, and pili
were named, no one could have predicted their relationships
to each other; this confusion is the result.
Regulation of competence in B. subtilis
It is achieved through a two-component regulatory system: a
sensor protein (ComP) and a response regulator (ComA) protein.
1. When the cell runs out of nutrients and the population reach a
high density registered by ComP.
2. ComP autophosphorylates itself.
3. ComP transfer ~P to ComA.
4. ComA~P is an active transcriptional activator for several genes,
including some required for competence.
5. Eventually, another transcriptional activator, ComK is made.
It is directly responsible for activating the transcription of other
com genes, including those that form the transformation
machinery.
Regulation of competence in B. subtilis
How does the cell know that other B. subtilis cells are
nearby and that it should induce competence?
1. High cell density is signaled through small peptides,
competence pheromones that are excreted by the bacteria as
they multiply.
2. Cells become competent only in the presence of high
concentrations of these peptides.
3. This is a phenomenon called quorum sensing. The small
molecules are known as including homoserine lactones that
signal cell density in some G – bacteria.
4. In B. subtilis, the major competence pheromone peptide is
called ComX and is cut out of a longer polypeptide, the product
of the comX gene.
Regulation of competence in B. subtilis
How does the cell know that other B. subtilis cells are
nearby and that it should induce competence?
5. The product of gene comQ which is immediately upstream of
comX is a protease that cut the longer polypeptide.
6. Once the peptide has been cut out of the longer molecule, it
binds to the ComP protein in the membrane and trigger its
autophosphorylation.
7. At best, only about 10% of B. subtilis cells ever become
competent, no matter how favorable the conditions or how high
the cell density. This has been called a bistable state and
seems to be determined somehow by autoregulation of the
ComK activator protein.
Regulation of competence development
in B. subtilis by quorum sensing
A
1. ComP in the membrane senses a high concentration
of the ComX peptide, and phosphorylates itself by
transferring a phosphate from ATP.
2. The phosphate is then transferred to ComA.
3. ComA activates the transcription of many genes
including comK.
4. ComK is an activator of the com genes.
B
1. In another pathway, a peptide sometimes called CSF
(competence-stimulating factor) processed from the signal
sequence of another protein (PhrC) is imported into the cell by
the SpoOK oligopeptide permease.
2. CFS indirectly activates ComA~P by inactivating RapC.
Regulation of competence development
in B. subtilis by quorum sensing
Relationship between competence,
sporulation, and other cellular states
1. About the same time as B. subtilis reaches the
stationary phase, some cells acquire competence
and some cells sporulate, forming the endospore.
2. Sporulation allows a bacterium toenter a dormant
state and survive adverse conditions, such as
starvation, irradiation and heat.
3. To coordinate sporulation and competence, B.
subtilis cells may produce other competence peptide.
(1) There are at least two such peptides that regulate
ComA indirectly by inhibiting proteins, Rap
proteins, which bind to the C-terminal DNAbinding domain of ComA~P and it from binding
to DNA and activating transcription.
Relationship between competence,
sporulation, and other cellular states
(2) These peptides (CSF) are processed from the signal
sequences of longer polypeptides, the products of
the phr genes, and are transported into cell by the
oligopeptide permease, SpoOK.
(3) The spoOK gene is an example of a regulatory gene
that is required for sporulation and also for the
development of competence.
Three questions for natural
transformation
A. How efficient is DNA uptake?
- Donor DNA is radioactively labeled by growing the cells in
medium containing 32P.
- The radioactive DNA is then extracted and mixed with
competent cells.
- The mixture is treated with DNase at various times.
- Any DNA that is not degraded and survives intact
must have been taken up by the cells, where it is protected
from the DNase.
- Collect cells on filter and count the radioactivity.
Degraded DNA will pass through filter.
- The radioactivity on the filter is compared with the total
radioactivity of the DNA that was added to the cell.
- This kind of experiment shows that some competent bacteria
take up DNA very efficiently.The % shows some
Efficient of DNA uptake
Three questions for natural
transformation
B. Can only DNA of the same species enter a given cell?
- The same experiment demonstrates that some types of
bacteria take up DNA from only their own species (ex.,
Neisseria gonorrhoeae and Haemophilus influenzae) whereas
others (B. subtilis) can take up DNA from any source.
- Bacteria that preferentially take up the DNA of their own
species do so because their DNA contains specific uptake
sequences.
-
Transformation in Streptococcus
pneumoniae
3. The remaining single
strand protected by a
DNA-binding protein
replaces the strand of
the same sequence in
bacterial chromosome,
creating a “heteroduplex”
in which one strand
comes from the donor
and one comes from the
recipient.
1. Competence-stimulating peptide accumulates as the cells
reach a high density.
2. Double-stranded DNA binds to the cell, and one strand is
degraded.
Transformation in Haemophilus
influenzae
3. The new DNA may not
become single
stranded until it
enters the cytoplasm.
Only one strand of the
DNA enters the
interior of the cell and
integrates with the
cellular DNA to
produce recombinant
types.
3. The basic transformation scheme may differ among different
types of naturally competent bacteria.
4. In H. influenzae, the double-stranded DNA may first take up in
subcellular compartments called transformsomes.
Three questions for natural
transformation
C. Are both of the DNA strands taken up and
incorporated into the cellular DNA?
- Experiments have shown that only doublestranded DNA can bind to specific receptors on the
cell surface, i.e., single-stranded DNA can not
transform cells and yield recombinant types.
- However, the transforming DNA enters a “eclipse”
period for a short time after it is added to
competent cells, as expected if it enters the cell in
a single-stranded state.
- The following is the design for experiment:
Whether both of the DNA strands taken
up and incorporated into the cellular DNA?
Whether both of the DNA strands taken
up and incorporated into the cellular DNA?
As shown in Fig. 6.8, transforms were observed depending on the
time the DNA was extracted from the cells.
1. Time 1, the DNA is still outside the cells and accessible to the
DNase. No Arg+ transformants are observed because the Arg+
donor DNA is all destryed by DNase.
2. Time 2, some of DNA is now inside the cells, where it can not
be degraded by the DNase, but this DNA is single-stranded.
It has not yet recombined with bacterial chromosomal DNA, and
so no Arg+ transformants observed in step 4.
3. Time 3, when some of the DNA has recombined with bacterial
chromosomal DNA, and so is again double-stranded, do
transformants appear in step 4.
■ Thus, the transformingf DNA enters the eclipse period for a
short time after it is added to competent cells, as expected if it
enters the cells in a single-stranded state.
Plasmid transformation and phage transfection
of naturally competent bacteria
Neither plasmids nor phage DNAs can be efficiently introduced
into naturally competent cells for two reasons:
1. They must double stranded to replicate. Natural transformation
requires breakage of double-stranded DNA and degradation of
one of the two strands so that a linear single strand can enter
the cells.
2. They must recyclize. However, pieces of plasmid or phage
DNA can not recyclize if there are no repeated or
complementary sequences at their ends.
- To overcome the problem, they are usually dimerized and
multimerized into long concatemers.
- If a dimerized plasmid or phage DNA is cut only once, it still
has complementary sequences at its ends that can
recombine to recylize the plasmid.
Plasmid transformation and phage transfection
of naturally competent bacteria
- Evidences to
support: Most
preparations of
plasmid or
phage DNAs
contain some
dimers.
- The fact that only dimerized plasmid or phage DNAs can
transform naturally competent bacteria supports the model of
uptake of single-stranded DNA during transformation.